1. Field
The present disclosure relates to compositions useful for treatment of retinal pathologies, including diabetic retinopathy, and to methods of making and using said compositions.
2. Description of Related Art
Diabetes mellitus is characterized by hyperglycemia (high glucose in the blood), which results either from insufficient production of insulin (type 1 diabetes) or from a cellular insensitivity to insulin in the blood. Diabetic retinopathy is a severe complication of diabetes, affecting the vision of more than half of adult diabetics, and is the leading cause of blindness in adults in the United States (1). The mechanisms of diabetic retinopathy and therapeutic strategies for treating it are the subject of extensive efforts. In a clinical setting, for example, laser photocoagulation and anti-angiogenic therapy represent state-of-the-art therapeutic strategies for inducing angiogenic regression and reduction of macular edema. Nevertheless, therapeutic challenges remain because many patients are unresponsive to current therapeutic approaches and/or because the state-of-the-art anti-angiogenic and photocoagulation therapies are accompanied by significant side-effects.
Diabetic hyperglycemia is thought to cause injury to endothelial cells that line retinal blood vessels, resulting in inflammation and neovascularization (growth of new blood vessels), which are characteristic of diabetic retinopathy. Extracellular matrix (ECM) is a cementing substance that supports and maintains the integrity of cells, including retinal endothelial cells. Hyperglycemia induces retinal endothelial cells to increase the production of ECM-degrading enzymes, including heparanase and matrix metalloproteinases (MMPs). Heparanase degrades the ECM by degrading heparan sulfate moieties into shorter-length oligosaccharides, while MMPs are zinc-dependent endopeptidases that also degrade ECM. Hyperglycemia-induced amplification of heparanase and MMP production results in inflammation and dysregulation of the blood-retinal barrier (BRB), leading to neovascularization and/or leakage of retinal blood vessels (diabetic macular edema). Strategies targeting ECM-degrading enzymes could be therapeutically beneficial for treatment of diabetic retinopathy, yet success has remained elusive.
Heparanase is implicated in angiogenesis, inflammation and metastasis (15). It is synthesized as a 65 kDa inactive precursor that undergoes proteolytic cleavage, yielding a 50 kDa active unit (9). Upregulated expression of heparanase was reported in vitro in human RE cells exposed to elevated levels of glucose, and in streptozotocin-induced diabetic rats in vivo. Heparanase inhibitors can be anti-angiogenic (9). A drug known as PI-88 (Phosphomannopentose sulfate-88) is currently under Phase II and III clinical trials known to function as a competitive inhibitor of heparanase. PI-88 is known to inhibit tumor angiogenesis and metastasis. However, dose-limiting toxicity of PI-88 is reported to inhibit thrombin-induced platelet aggregation and to prolong anti-coagulant activities. Heparanase is known to play many important roles in the regulation of several aspects of cancer biology, including angiogenesis, tumor progression, and metastasis (9). Heparanase is an endo-β-D-glucuronidase that degrades HS-proteoglycans (HSPG) in the extracellular matrix (EM) and the basement membrane. The angiogenic capacity of heparanase has been traditionally attributed to its ability to release HS-bound angiogenic growth factors from the ECM, such as vascular endothelial growth factor (VEGF). The use of HS mimetics to modulate these processes may therefore present a promising approach for ECM degradation-based therapies.
Matrix metalloproteinases (MMPs) are zinc-dependent ECM degrading enzymes (12). MMP regulates BRB functions of several tissues including those in the eyes. Hypergycemic injury and laser induced injury reported to elevate the expression of MMPs in the posterior eye. The increased proteolytic activity of ECM degrading enzymes facilitates the permeability changes of BRB. An inhibitor of MMPs was able to inhibit the diabetic hypoglycemia-induced BRB breakdown (12).
In the eye, various matrix-degrading enzymes including, for example, endoglycosidases and matrix metalloproteinases are thought to cause remodeling of the tight structural extracellular matrix (ECM) and basement membrane (BM) networks in response to altered microenvironments of BRB compositions. The BRB is formed by retinal endothelilal cells, and is essential for protecting the retina from harmful agents found in the blood. Retinal endothelial cells form tight junctions (TJs) that are essential to maintaining the structural integrity of the BRB. Degradation of the BRB, which may be influenced by the extracellular matrix degradation, is known to result from various causes including, for example, hyperglycemic injury or laser treatment of proliferative retinal angiogenesis (8).
ECM degradation of BRB is known to result from hyperglycemic injury or laser treatment of proliferative retinal angiogenesis. Loss of BRB integrity and leakage of plasma constituents leads to vision loss and associated microvascular complications of the posterior eye. Besides affecting the tight junction proteins, expression and activity of specific extracellular proteinases is known to change the endothelial permeability of BRB.
Impairment of the BRB tight junctions can occur through the actions of various matrix-degrading enzymes including, for example, endoglycosidases and matrix metalloproteinases (MMPs).
Endoglycosidases contribute to releasing sequestered heparan sulfate-binding proteins (e.g., VEGF and other growth factors), which are then free to signal through their cognate receptors, leading to alterations in the ECM that modulate BRB permeability and may facilitate angiogenesis. One such matrix-degrading enzyme is heparanase, an endoglycosidase that specifically cleaves the heparan sulfate (HS) side chains of HSPGs (9). Heparanase has been implicated in human cancer, particularly in malignant, aggressive tumors (9). These data have come largely from correlative studies documenting a positive association between increased heparanase expression or activity and enhanced tumor invasion and metastasis (9). The ECM remodeling role of heparanase was confirmed in several in vitro and in vivo model systems, including wound healing (9), tumor xenografts (10), Matrigel plug assay (10), and tubelike structure formation (11). Heparanase offers an attractive drug target. Species of heparin and heparin/HS-mimicking compounds that inhibit the enzyme may prevent undesirable vascular remodeling. The matrix metalloproteinase's (MMPs) are zinc-dependent ECM-derading enzymes. MMPs regulate the functions of several tissues including those in the eyes (12) including, for example, the BRB.
Apolipoprotein E (apoE) is a protein that was first recognized for its importance in the metabolism of lipoproteins and its role in cardiovascular disease. Subsequent research has demonstrated the involvement of apoE in various biological processes including, for example, Alzheimer's disease, immunoregulation, and cognition. Human apoE is initially synthesized as a propeptide of 317 amino acids. Following post-translational cleavage of an 18 amino acid (“a.a.”) signal peptide, mature apoE is secreted as a 34.2 kDa protein consisting of 299 amino acids. ApoE is a single-chain protein containing two independently folded functional domains—a 22-kDa N-terminal domain (a.a. residues 1-191) and a 10-kDa C-terminal domain (a.a. residues 222-299)—and is a ligand for cell-surface heparan sulfate proteoglycan (HSPG) (2,3). The N- and C-terminal domains of apoE each contain a heparin binding site (13). The N-terminal domain heparin binding site is located between residues 142-147, within the apoE heparan sulfate binding region (see SEQ ID NO: 2), and overlaps the receptor binding region of SEQ ID NO:1 (3). In fact, the HSPG binding activity of apoE variants is significantly decreased by mutations of Arg-142, Arg-145, and Lys-146, indicating that these basic amino acid residues contribute to binding of both heparin and heparan sulfate proteoglycan (4). A tandem repeat dimer peptide derived from apoE residues 141-149 reportedly bears anti-inflammatory and anti-angiogenic activity in vivo against herpes virus infection (5); the tandem repeat may reflect increased adoption of α-helical structure and improved stability (6). ApoE is expressed in almost all cells, including retinal endothelial (RE) cells, and the degree of apoE expression in the retina is almost equal to that observed in the brain (7).
Treatment with a human apolipoprotein E derived dimer peptide (apoEdp) blocks VEGF-induced ocular angiogenesis in a rabbit eye model (17). The heparan sulfate (HS) binding domain of human apolipoprotein E (apoE) possesses anti-tumorigenic and anti-angiogenic roles through inhibition of vascular endothelial growth factor (VEGF), but whether this peptide has a vascular remodeling role through inhibition of ECM degrading enzyme heparanase is unknown.
The solution to these technical problems is provided by the embodiments characterized in the claims.